Water-soluble cholesterol derivative

ABSTRACT

Complexes of albumin combined with organic dibasic acid half esters, such as those of succinic acid and phthalic acid, of 7-hydroxycholesterol are soluble in water and have excellent immunosuppressive and anti-inflammatory action.

TECHNICAL FIELD

The present invention relates to water-soluble derivatives ofcholesterol and more particularly to water-soluble derivatives of7-hydroxycholesterol.

BACKGROUND ART

It is known that the immunoregulatory substance isolated from the CohnIV, paste of human serum is 7-hydroxycholesterol and effective not onlyas an immunoregulatory agent, particularly acting to regulatecell-mediated immunity, but also as an anti-inflammatory agent (JapanesePatent Application Laid-open No. 104735/1978).

DISCLOSURE OF INVENTION

Since 7-hydroxycholesterol, a compound very valuable as a medicine, issparingly soluble in water, the present inventors made intent studiesaiming at development of its derivatives having improved watersolubility.

As a result, the aim has been attained by finding out new compounds,complexes of albumin with organic dibasic acid half esters of7-hydroxycholesterol.

Thus, the object of this invention is to provide said complexes, whichare represented by the formula ##STR1## wherein R is phenylene or(CH₂)_(m), of which m indicates 0 or an integer of 1-5, P is albuminresidue, and n is a number of 20-200, which indicates the number ofmoles of the steroid, shown in the bracket, binding to mole of albumin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show results of gel filtration of the respective compoundsobtained by reacting 1 part of albumin and 3 parts of albumin,respectively, to 1 part of 7-hydroxycholesterol-3-succinic acid halfester.

In these figures, concentrations of albumin are represented by measuredvalues of the O.D. (optical density) at 280 nm ( ), concentrations of7-hydroxycholesterol-3-succinic acid half ester are represented bymeasured values of the O.D. at 640 nm ( ) after development of color bythe Lubschutz reaction, and peak A, peak B, and V_(o) correspond to thecomplex, the unreacted albumin, and the void volume, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

An organic dibasic acid half ester of 7-hydroxycholesterol, used as astarting material in this invention, which is represented by the formula##STR2## wherein R is as defined above, can be prepared from7-ketocholesterol by esterifying it with a reactive derivative of thecorresponding organic dibasic acid in the presence of a tertiary amine,followed by the reduction with a complex metal hydride (Japanese PatentApplication No. 7676/1979). Preferred organic dibasic acids used includesuccinic acid and phthalic acid, and preferred starting materialsinclude 7-hydroxycholesterol-3-succinic acid half ester,7-hydroxycholesterol-3-phthalic acid half ester, and7β-hydroxycholesterol-3-phthalic acid half ester.

The compounds of this invention are conveniently produced by uniformreaction of half esters above mentioned with albumin in a mixture ofwater and a water-misible organic solvent such as dioxane,tetrahydrofuran, or dimethylsulfoxide. Water is a good solvent foralbumin and the organic solvent is so for the half ester. Thus, thereaction is carried out in such a way that such an organic solvent isadded to an aqueous solution of albumin and then a solution of the halfester in the organic solvent is added to the resulting mixture withstirring or in the reverse addition order. Organic solvent to waterratios allowing enough uniform progress of the reaction are acceptable,which are 1:1-10.

The albumin to be used may be of human or bovine serum, and it is notparticularly restricted as far as purified for therapeutic purposes.Needless to say, those of human derivation are preferred. The purity ofthe albumin is preferably not less than 80% as measured by theelectrophoretic method. The amount of albumin used for preparation ischosen from within the range of 0.2-10 parts by weight for part byweight of the organic dibasic acid half ester of 7-hydroxycholesterol,so that the complex having a desired binding ratio can be obtained. Anincrease in the proportion of albumin makes the water-solubility of thehalf ester increased, but this is unfavorable with respect totherapeutic efficacy since it decreases the half ester content at thesame time. It is more favorable to bind a maximum possible amount of thehalf ester to one mole of the albumin. The binding molar ratio is 20-200moles, preferably 50 moles of more, of half ester to mole of albumin. Inorder to increase the binding ratio, the amount of half ester added maybe increased, thereby a desired result being obtained.

The reaction temperature, though not particularly limited, is usually0°-10° C. In consideration of the solubility of albumin, the reaction isdesirable to be carried out in an alkaline region. Accordingly, forinstance, a small amount of NaOH is added to the reaction system toadjust the pH to 8-10. A reaction time of 1-5 hours is adequate. Alonger reaction time has no particular effect.

The reaction liquid obtained is freed from unreacted reactants asrequired. For removing them, known techniques can be applied. Apreferred technique for this purpose is gel filtration, wherein thecarrier to be used is not particularly restricted as far as suited tothe separation of protein. A wide variety of gel filtration carriersfitted to the molecular weight of serum albumin (about 64,900) can beutilized. As examples thereof, there may be cited Sephadex (crosslinkeddextran) G-200, G-150, and G-100 and Sepharose (agarose) 4B and 6B(supplied by Pharmacia, Ltd., Sweden).

The unreacted albumin and half ester may be incorporated with thecomplex in preparations without separation. In particular, free albuminhas a stabilizing effect on preparations when contained therein insuitable amounts (0.01-10 wt%).

The water-soluble derivative of cholesterol thus obtained is then madeup to preparations according to a desired known preparation method. Forinstance, the resulting liquid is then dialyzed against a buffersolution to be desalted, and the desalted inner liquid is recovered,subjected to sterilizing filtration, if necessary, and lyophilized todry preparations. The buffer solution used in the dialysis is preferablyadjusted to pH 7.0-8.0. For example, phosphate buffer, citrate buffer,and acetate buffer can be used for this purpose. The ionic concentrationis 0.001-0.1 M.

The preparations of this invention may contain, as desired, knownadjuvants such as carrier, excipient, dissolving aid, and stabilizer,which are added according to conventional methods. A preferred form ofthe preparations in application thereof as a therapeutic agent is aninjectable one, and the dose in this case is 10-1000 mg/Kg as organicdibasic acid half ester of 7-hydroxycholesterol. When applied as aninjection medicine, the present preparations are preferably dissolvedeach time in water to use as an 0.1-20% aqueous solution. The presentpreparations are, of course, available as medicines for externalapplication and for oral application.

The complexes thus obtained of albumin with organic dibasic acid halfesters of 7-hydroxycholesterol are new compounds unreported in theliterature, and exhibit excellent efficacy in immunoregulation,especially in suppression of cel-mediated immunity and inanti-inflammation. Moreover, they readily dissolve in water forming aclear, stable, and neutral solution, and are therefore very useful aspharmaceuticals.

Experiment data on the usefulness of the compound of this invention willbe shown below which were prepared in the following Example 1 but notyet subjected to gel filtration.

(1) Immunoregulatory action (tests in vitro)

The 50% inhibitory concentration of the compound of this invention inthe PHA reaction according to S. R. Cooparband et al. [The Journal ofImmunology, 109, No. 1,154 (1972)] was tested. The result is shown inTable 1. Additionally, the result of the same test on theimmunoregulatory α-globulin (IRA) obtained by the method of Occhino etal. [The Journal of Immunology, 110, (3) (1973)] is shown forcomparison.

                  TABLE 1                                                         ______________________________________                                                         50% inhibitory concentra-                                                     tion in the PHR reaction                                     Sample           (μg/ml)                                                   ______________________________________                                        Complex of albumin with                                                                         3                                                           7-hydroxycholesterol-3-                                                                        (as steroid)                                                 succinic acid half ester                                                      IRA              20                                                           ______________________________________                                    

(2) Immunoregulatory action (tests in vivo)

Animal tests were carried out using five mice for each sample. Anaqueous solution of the complex of albumin with7-hydroxycholesterol-3-succinic acid half ester (8 mg/Kg/day assteroid), an aqueous solution of albumin (8 mg/Kg/day), and saline as acontrol were intravenously injected into mice during 20 days insuccession to examine the effect of each sample on the transplantationof skin. The mode of administration was as follows: to each rat a dosewas given 24 hours before the skin transplantation and thereafter theadministration of a dose per day was continued for 20 days. According tothe 20-days later observation of surface, the percentage of successfulgrafts was 0% for the control, about 5% for the albumin, and about80-90% for the complex of this invention. This indicates pharmacologicaltherapeutic effectiveness of the complex as an immunoregulatory agent.

(3) Anti-inflammatory action

Animal tests were carried out using five rats for each sample. Anaqueous solution of the complex of albumin with7-hydroxycholesterol-3-succinic acid half ester was injected into theabdominal cavity of each rat (50 mg/Kg as steroid) and 1 hour later 0.05ml of 1% aqueous carrageenin was hypodermically inoculated as aninflammatory agent to a hind leg of each rat. Four hours afterinoculation of carrageenin, the volume of the edema formed on the legwas measured by means of a volume differential meter, and on the volumeof edema the complex was compared with the reference sample albumin (adose: 50 mg/Kg) and with the control saline. The results are shown inTable 2.

                  TABLE 2                                                         ______________________________________                                                          Increase in volume                                          Sample            of edema (%)                                                ______________________________________                                        Saline            82                                                          Albumin           78                                                          Complex of albumin with                                                                         28                                                          7-hydroxycholesterol-                                                         3-succinic acid half                                                          ester                                                                         ______________________________________                                    

(4) Acute toxicity test

Acute toxicity was tested on the present compound using each group offive rats each weighing about 200 g. The compound in the form of aqueoussolution was abdominally injected in doses of 250, 750, and 1000 mg/Kgas weight of steroid for respective groups of rat. As a result of fivedays observation, no case of death was found.

Thus, the compound of this invention is almost nontoxic and notantigenic so that it can be provided as an excellent immunoregulatoryagent or anti-inflammatory agent.

The present invention will be illustrated by the following referenceexample of preparation of starting material and examples of carrying outthe invention.

Reference Example 1 (preparation of starting material)

(1) After 5 g of 7-ketocholesterol was dissolved in 50 ml pyridine, 1.8g of succinic anhydride was added thereto, the mixture was heated toreact for 8 hours at 90° C. and allowed to stand at room temperatureovernight. Then, the resulting liquid was poured into 500 ml ice-coldwater. The crystals precipitated by acidification with hydrochloric acidwere collected by filtration and then dissolved in ethanol, activecarbon was added thereto for decoloration and filtered off, water wasadded to the filtrate, and the precipitated crystals were recovered byfiltration. Upon recrystallization thereof from aqueous ethanol, 5.4 gof 7-ketocholesterol succinic acid half ester was obtained as flakycrystals, m.p. 187.5°-189.5° C.

Elemental analysis:

Calcd. for C₃₁ H₄₈ O₅ : C, 74.36; H, 9.66, Found: C, 74.54; H, 9.59.

I.R. absorption spectrum (KBr tablet): 1735, 1705, 1670, 1170 cm⁻¹.

(2) While stirring, 820 mg sodium borohydride was slowly added to asolution of 3.75 g sodium salt of 7-ketocholesterol succinic acid halfester in 100 ml methanol under cooling with ice. Stirring was continuedfor 10-15 minutes with ice-cooling and for further 1 hour at roomtemperature. The resulting liquid, after excess sodium borohydride wasdecomposed by adding 2 ml acetic acid, was diluted with 150 ml water andacidified with hydrochloric acid. Precipitated crystals were filteredand recrystallized from cyclohexane. Thus, 3.43 g of7-hydroxycholesterol-3-succinic acid half ester was obtained asneedle-like crystals, m.p. 150°-154° C. This product was a mixture of7α-hydroxy and 7β-hydroxy forms in a ratio of about 1:3, the latterbeing major.

Elemental analysis:

Calcd. for C₃₁ H₅₀ O₅ : C, 74.06; H, 10.02, Found: C, 74.21; H, 9.96.

I.R. absorption spectrum (KBr tablet): 3500, 1730, 1705, 1165 cm⁻¹.

EXAMPLE 1

A solution of 900 mg human serum albumin in a mixture of 60 ml water,0.9 ml 1 N NaOH, and 30 ml dioxane was slowly added to a solution of 900mg 7-hydroxycholesterol-3-succinic acid half ester in 21 ml dioxanewhile stirring at 5°-10° C., and stirring was continued for 3 hours.Thereafter the resulting liquid was dialyzed against 0.05 M phosphatebuffer (pH 7.4) at 5°-10° C. for 24 hours. Upon lyophilization of thedialized inner solution in vacuo, 2.9 g of a white dry powder wasobtained.

Physicochemical properties of this product are as follows:

Herein the 7-hydroxycholesterol-3-succinic acid half ester constituentwas determined by gas chromatography through the alkali hydrolysis into7-hydroxycholesterol followed by tetramethylsilyl substitution in theknown way, and the albumin constituent was determined by the Lowrymethod.

(1) Composition:

7-hydroxycholesterol-3-succinic acid half ester: 29.14%

Albumin: 33.10%.

Phosphate: 37.76%.

(2) Binding steroid: 117.5 mol/mol of albumin,

(3) Solubility: 200 mg/ml of H₂ O,

(4) pH: 7.4 (2% aqueous solution).

Results of the gel filtration through Sephadex G-200 are shown in FIG.1.

While this product showed its enhanced usefulness in the foregoinganimal tests, 2 g of a further purified product was obtained therefromby gel filtration. Conditions of the gel filtration were as follows:

Gel filtration carrier: Sephadex G-200

Column: K 16/70,

Bed capacity: 120 ml (1.6×60 cm),

Eluent: 1/15 M phosphate buffer, pH 7.4, (containing 0.01% of NaN₃),

Flow rate: 6.0-6.5 ml/hr,

Fraction: 3.0 ml/tube.

EXAMPLE 2

A solution of 2.7 g human serum albumin in a mixture of 180 ml water,2.7 ml 1 N NaOH, and 90 ml dioxane was slowly added to a solution of 900mg 7-hydroxycholesterol-3-succinic acid half ester in 21 ml dioxanewhile stirring at 5°-10° C., and stirring was continued for 3 hours.Thereafter, in the same manner as in Example 1, 6.8 g of a dry powderwas obtained.

Physicochemical properties of this product are as follows:

(1) Composition:

7-hydroxycholesterol-3-succinic acid half ester: 12.03%,

Albumin: 41.31%,

Phosphate: 46.66%,

(2) Binding steroid: 38.9 mol/mol of albumin,

(3) Solubility: 250 mg/ml of H₂ O,

(4) pH: 7.4 (2% aqueous solution).

By the same gel filtration procedure as in Example 1, 6 g of a purifiedproduct was obtained. Results of the gel filtration are shown in FIG. 2.

We claim:
 1. A complex of albumin with an organic dibasic acid halfester of 7-hydroxycholesterol, said complex being represented by theformula ##STR3## wherein R is phenylene or (CH₂)_(m), of which mindicates 0 or an integer of 1-5, P is albumin residue, and n is anumber of 20-200 indicating the number of moles of the steroid, shown inthe bracket, binding to mole of albumin.
 2. A process for preparing acomplex of albumin with an organic acid half ester of7-hydroxycholesterol, said complex being represented by the formula##STR4## wherein R is phenylene or (CH₂)_(m), of which m indicates 0 oran integer of 1-5, P is albumin residue, and n is a number of 20-200indicating the number of moles of the steroid, shown in the bracket,binding to mole of albumin, said process comprising reacting albuminwith a dibasic acid half ester of 7-hydroxycholesterol represented bythe formula ##STR5## wherein R is as defined above, and recovering thereaction product.
 3. A process according to claim 2, wherein 0.2-10parts by weight of albumin is used per part by weight of the organicdibasic acid half ester of 7-hydroxycholesterol.
 4. A process accordingto claim 2, wherein the reaction is carried out in a mixed medium ofwater and a water-miscible organic solvent.
 5. A process according toclaim 2, wherein the reaction is carried out using 20-200 moles of theorganic dibasic acid half ester of 7-hydroxycholesterol per mole ofalbumin.
 6. A process according to claim 2, wherein the recovery ofreaction product is carried out by gel filtration to separate from thefree albumin.
 7. A composition for transplantation of organ, containinga water-soluble complex of claim 1 of albumin with an organic dibasicacid half ester of 7-hydroxycholesterol.
 8. A composition for treatinginflammation, containing a water-soluble complex of claim 1 of albuminwith an organic dibasic acid half ester of 7-hydroxycholesterol.
 9. Usefor transplantation of organ of a water-soluble complex of claim 1 ofalbumin with an organic dibasic acid half ester of 7-hydroxycholesterol.10. Use for treating inflammation of a water-soluble complex of claim 1of albumin with an organic dibasic acid half ester of7-hydroxycholesterol.